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Abstract

Influenza A virus (IAV) is a significant cause of respiratory infections globally, with thousands of casualties annually. Its capacity for rapid antigenic variation in the surface glycoproteins, hemagglutinin (HA), and neuraminidase (NA) leads to the emergence of novel viral subtypes, thereby undermining vaccine efficacy and reinforcing its clinical relevance. During viral budding in polarized cells, IAV utilizes lipid raFs - membrane microdomains enriched in cholesterol and sphingolipids (SLs) - at the apical side of the cell membrane, resulting in a viral envelope enriched in these lipids. HA and NA, are characterized as raft-associated proteins, which are likely recruited to these domains during viral assembly. The NA1 protein, in particular, might possess an SL-interac9ng motif, potentially aiding its localization to the budding site. Additionally, the number of SLs increases during IAV infection, and defects in sphingomyelin (SM) synthesis affect the localization of viral glycoproteins to the plasma membrane. Despite the established role of SLs in IAV infection, the precise molecular mechanisms by which specific SLs contribute to viral propagation, or how IAV modulates the SL metabolic pathway, remain elusive. The involvement of SL-binding host proteins in viral replication has not yet been thoroughly explored. To address this, a stable isotope labeling by amino acids in cell culture (SILAC) proteomics approach was employed, using bifunctional sphingosine to identify SL-interacting proteins in infected and non-infected A549 cells (AG Brügger). Several SL-interacting protein hits were selected for further investigation in IAV infection based on their functional relevance and existing literature evidence of their roles in various viral infections. Knockdown experiments assessed the impact of several candidate proteins on IAV infection. Among these, ceramide synthase 2 (CerS2) was identified as a key protein with potential antiviral effect. CerS2 catalyzes the transfer of an acyl chain from acyl-CoA to a sphingoid base, with a high selectivity for very long-chain fatty acids. Infection of two A549ΔCerS2 clones with the PR8 strain of IAV displayed increased levels of viral nucleoprotein (NP) expression and plaque titers, with some clonal differences in HA titer. Notably, the reexpression of CerS2 in the A549ΔCerS2 cells led to a significant reduction in NP expression, HA titers, and plaque formation, supporting the hypothesis of an antiviral function for CerS2 in IAV infection. However, the molecular mechanisms through which CerS2 influences IAV infection remain to be elucidated in future studies. In summary, this study provides novel insights into the role of sphingolipid-interacting proteins, particularly CerS2, in the replication and pathogenesis of IAV. These findings open new avenues for future research and may contribute to the development of innovative therapeutic strategies aimed at combating IAV infection